CLINICAL BACTERIOLOGY (LECTURE)

LESSON 7: STREPTOCOCCUS AND ENTEROCOCCUS

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2nd SEMESTER I S.Y. 2021-2022
TRANCRIBED BY: JEAN HERSHEY REYES

General Characteristics colony induced by bacterial hemolysins by bacterial

hemolysins
 Streptococcus and Enterococcus spp. o No red blood cell is visible on microscopic examination in
belong to the family Streptococcaceae clear zone of complete hemolysis
 Inhabit various sites, notably the upper
respiratory tract and live harmlessly as  ß-hemolysis constitutes the principal marker for potentially
commensals. principal marker for potentially pathogenic streptococci pathogenic
streptococci in in cultures of throat swabs or cultures of throat
 Both of the genera are catalase-negative
swabs or other clinical samples.
(weak reaction), gram-positive cocci that
are usually arranged in pairs or chains
C. Gamma (γ) or Non-hemolytic Streptococci
 The cells of Enterococci and some
Streptococci appear more elongated than
spherical  Produce no hemolysis on blood agar.
 The streptococcal cells are more likely to  Enterococcus faecalis is an important organism of this group.
appear in chains when grown in broth
cultures Alpha Beta Gamma
 Most members are aerotolerant anaerobes Color around Green Clear Red
(but behave like facultative anaerobes) colonies
 Some species are capnophilic Members S. pneumoniae S. equisimilis Some
S. pyogenes Viridans
 Poor growth on Nutrient Media such as Trypticase Soy Agar S. agalactiae group
 More pronounced growth on media enriched with blood or serum E. faealis
 Colonies are usually small and transparent
 Most Streptococci, have a group or common C carbohydrate  S. pneumoniae, S. pyogenes, S. agalactiae, E. faealis – true
(polysaccharide) which can be used to classify an isolate pathogens
serologically (Lancefield classification)
Blood Agar:
o Rebecca Lancefield
Shows three types of hemolysis
Classification of Streptococcus spp. 1. Alpha-hemolysis
2. Beta-hemolysis
3. Gamma-hemolysis
Academic/Bergy Smith and Lancefield
Brown
Basis of Temperature Hemolysis Serological
Classification (C-carb of
cell wall)
Members Pyogenic Alpha Groups A-G
Viridance Beta
Entero Gamma
Lactic
Lancefield Classification (Serological Properties)
Smith and Brown Classification
Lancefield Grouping
A. Alpha-hemolytic (α) Streptococci
 It was developed by Rebecca Lancefield in the year 1930s
 Produces a zone of partial  Designated by letters
hemolysis with a greenish o Groups A, B, C, D, and G are most and G are most
discoloration around the commonly found commonly found associated with
colonies on blood agar associated with human infections.
 Streptococci producing α-
hemolysis are also known as  Basis of group specific carbohydrates (c) antigens in the cell wall
Viridans Streptococci.
Group A Group B Group C Group D
B. Beta (ß) Hemolytic Streptococci
S. pyogenes S. agalactiae S. equisimilis A. Enterococci
 Produces a complete hemolysis S. equi
o Sharply defined, clear, S.dysogalactiae E. faecalis
Sharply defined, clear, S. zoopidimidus E. faecium
colorless zone of E. avium
hemolysis colorless zone E. durans
of hemolysis around the

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 B. Non-  Staphylococcus is Catalase
Enterococci Negative (Dark)

S. bovis D. Bacitracin o Differentiates Group A Streptococci from

S. equinus Susceptibility other Streptococci
Test (Taxo A)  Positive: Group A Strep
Academic or Bergy’s Classification  Negative: Other Beta-hemolytic
Streptococci spp.
10 C 37 C 45 C Members
Pyogenic - + - Streptococc E. CAMP Test o Test for Group B Streptococci
us pyogenes (Christie Atkins o Known organism (S. aureus)
Viridance - + + S. mutans Munch Petersen o Unknown organism [Beta-hemolytic,
S. salivarius Test) Catalase (-), pinpoint, Bacitracin Resistant]
S. mitis  Group B Strep
S. bovis  S. agalactiae
S.
anginosus o Positive: arrowhead hemolysis [Group B
Entero + + + Enterococcu Strep] or bowtie appearance [S. agalactiae]
s faecalis o Christie, Atkinson, Munch, and Peterson
Lactic + + - Streptococc
us faecalis F. PYR o Positive: Cherry Red/Pink
Hydrolysis Test o Negative: Yellow
o Test for Group A and Group D Streptococci
Virulence Factors of Streptococci spp.
o Substrate: L-pyrrolidonyl-beta-napthylamide
o Reagent: p-dimethyl-aminocinnamaldehyde
 M Protein – S. Pyogenes – Genes emm o Enzyme: pyrroglutamylamino peptidase or
pyrrolidomylaryl amidase
Streptolysin S Streptolysin O
Type of hemolysin Surface of RBC Subsurface G. Hippurate o Test for Group B Streptococci
Oxygen Stable Labile Test o Medium: Broth with hippurate
Antigenicity Non-antigenic Antigenic o Reagent Indicator: Ninhydrin
o Hippurate  Benzoic Acid + Glycine
 M protein – part of cell wall; antiphagocytic o Add indicator after incubation
 Capsule – principal virulence factor of Group B Streptococci; o Positive: Purple (S. agalactiae)
antiphagocytic o Hippuricase
 Hemolysin
o Streptolysin O (oxygen labile) – highly immunogenic; H. Bile Esculin o Group D (Enterococci/Non-Enterococci)
measured using ASO (antistreptolysin O) test Test o Differentiate Group D from Beta-hemolytic
o Streptolysin S (oxygen stable) – nonimmunogenic o Enzyme: Esculinase
o Positive: blackening of medium [Group D]
 SpeA, SpeB, SpeC, and SpeF o ESCULIN  ESCULETIN

I. Salt o Further test from Bile Esculin Test

Toxin o Streptococcal pyrogenic exotoxin – formerly Tolerance Test o To identify Enterococci
called erythrogenic toxin; causes Scarlet (6.5% NaCl) o Negative: Clear/Transparent [Non-
Fever enterococci]
o Nephrogenic toxin – causes Acute o Positive: Turbid [Enterococci]
Glomerulonephritis
Streptokinase o spreading factors localized in the skin J. Leucine o Substrate: Leucine-β-naphthylamide  β-
Hyaluronidase o spreading factors localized in the skin Aminopeptidase o naphthylamine
Test o Reagent: paradimethylaminocin
DNAse o spreading factors localized in the skin; S.
namaldehyde reagent
pyogenes DNases: A, B (most common), C,
o Positive color: Red color
D
o Positive: Viridans Streptococci
Lipoteichoic acid o adhesion molecules
and Protein F
J. SXT o R – Group A & B Strep
CAMP Factor o Produced by S. agalactiae; heat stable
Susceptibility o S – Other Strep spp.
protein; enhances the beta-hemolysis of S.
Test
aureus
K. o Detect carbohydrate component of the cell
Serological wall of Streptococci
Laboratory Tests for Streptococcus spp. Tests o Name of test: Streptex
o Principle: Antigen(from colonies)-
Antibody(from Reagent) Reaction
A. Gram Stain o Gram-positive cocci in pairs or chains
B. Cultural o Pinpoint colonies Tube:
Characteristics  S. pyogenes – small, transparent  Add diluent (PBS/NSS) [0.5mL]
and smooth; beta-hemolytic  Transfer bacteria
 S. agalactiae – grayish white  Transfer to test card labelled A-G
mucoid colonies; beta-hemolytic  Add antibody
(small zone)
o Positive: agglutination/clumping [Group A
C. Catalase Test o Negative (no effervescence) Strep]
 Staphylococcus is Catalase o Negative: no agglutination [S. agalactiae]
Positive (bubbles)
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 o Functions as super antigen leading to overstimulation of
A B C D the immune response
Bacitracin S R R R  Initial streptococcal infection is severe (e.g., pharyngitis, peritonitis,
SXT R R S S cellulitis, wound infections)
CAMP - + - - o Then it develops into staphylococcal TSS-like symptoms
PYR + - - +
Sodium - - - - E. Poststreptococcal Sequelae
Hippurate
Bile - - - + Two serious complications or sequelae of GAS disease:
Esculin o Rheumatic Fever
6.5 NaCl - - - Entero  Follows after S. pyogenes pharyngitis
(+)  Characterized by fever and inflammation of the
Non- heart, joints, blood vessels and subcutaneous
entero (– tissues
)  Most serious result: chronic, progressive
damage to the heart valves
Clinically Significant Streptococci and Streptococcus-like
organisms o Acute Glomerulonephritis
 Follows after cutaneous or pharyngeal infection
 More common in children than in adults
Clinical Infections of S. pyogenes GAS (Group A Streptococcal)
Infection
 GAS are susceptible to penicillin (drug of choice for treatment)
 If allergic to penicillin – erythromycin
A. Bacterial Pharyngitis
Clinical Infections of Other Streptococcus spp.
 “Strep throat” – most often seen in children between 5 and 15
years of age
Streptococcus agalactiae
 Spread by droplets and close contact
 1-4 days incubation period
o Sore throat  Todd-Hewith Broth
o Malaise
o Fever Significant cause of invasive disease in newborn
o Headache o Most infections of infants occur in the first 3 days after
o Nausea, vomiting and abdominal pain (unusual) birth, usually within 24 hours
o Tonsils and pharynx are inflamed
o Cervical lymph nodes are swollen and Two clinical syndromes are associated with neonatal GBS disease:
early-onset infection (<7 days old) and late-onset infection (at least 7
days old to about 3 months old)
B. Pyodermal Infections
o Early-onset infection – pneumonia and sepsis
o Late-onset infection – meningitis and sepsis
1. Impetigo - a localized skin disease, begins as small vesicles that
progress to weeping lesions; inoculation of organisms through
 Commonly associated with obstetric complications, prolonged
minor abrasions or insect bites
rupture of membranes, and premature birth
 It is recommended that all pregnant women be screened for GBS
2. Cellulitis – followed by deeper invasion of streptococci; life-
at 35 to 37 weeks’ gestation
threatening; with bacteremia or sepsis
o Patients with peripheral vascular disease or diabetes –  In adults, the infection affects two types of patients:
o Young, previously healthy woman who become ill after
leads to gangrene
childbirth or abortion
3. Erysipelas - is a rare infection of the skin and subcutaneous  Endometritis and wound infections
tissues observed frequently in elderly patients
o Elderly person with a serious underlying disease or
o Lesion characteristics: acute spreading, intensely
immunodeficiency
erythematous with plainly demarcated but irregular edge

4. Scarlet Fever – cause by streptococcal pyrogenic exotoxin  Drug of choice: Penicillin

o Diffuse red rash on upper chest and spreads to the trunk o Some clinicians recommend a combination of ampicillin
and and aminoglycosides

C. Necrotizing Fasciitis

 “Flesh-eating disease or syndrome”, “Suppurative fasciitis”,

“Hospital gangrene”, “Necrotizing erysipelas”
 An invasive infection characterized by rapidly progressing
inflammation and necrosis of the skin, subcutaneous fat, and
fascia

D. Streptococcal Toxic Shock Syndrome

 A condition in which the entire organ system collapses, leading to

death
 Portal of infection is unknown, although minor injuries or surgeries
have been implicated
 Caused by a type of streptococcal pyrogenic exotoxin – SpeA
o Has a major role in the pathogenesis

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 Group C and G Streptococci

 S. equi subsp. zooepidemicus

Subdivisions:
o Large-colony forms
 Classified with the pyogenic streptococci
 Beta-hemolytic isolates – belong to the
subspecies S. dysagalactiae subsp. equisimilis
(also exhibited group A and L antigens)

o Small-colony forms
 Beta-hemolytic isolates – belong to the S.  Culture
anginosus group (Under viridans group) o BHIA, TSA with 5% sheep RBCs or Chocolate Agar are
necessary for good growth
Streptococcus pneumoniae o SBA – large zone of alpha-hemolysis

 Also known as pneumococcus or diplococcus  Cultural Characteristics

o Young cultures – round, glistening, wet, mucoid, dome-
 Member of S. mitis group
shaped appearance
 No Lancefield Classification
o Old cultures – coin with a raised rim appearance
o Antigen present in the cell wall: C substance
o C-reactive protein (CRP) reacts with C substance to form
 The colonies may closely resemble colonies of the viridans
a precipitate in human serum
streptococci
 Gram-positive cocci in pairs
 Optochin Test or Taxo P
 Alpha-hemolytic
o Chemical composed of ethylhydrocupreine hydrochloride
 Dome-shaped colonies o S = Pneumococci
o R = Viridans
Clinical Infections o Streptococci

 Lobar Pneumonia  Bile Solubility Test

 It is an important human pathogen that causes pneumonia, o For S. pneumoniae only
sinusitis, otitis media, bacteremia, and meningitis o Determines the lysis of S. pneumoniae in the presence of
 Most frequently isolate in children younger than 3 years old with bile salts
recurrent otitis media
 No. 1 cause of bacterial pneumonia – prevalent in elderly persons  Inulin Fermentation
and in patients with underlying conditions o Only S. pneumoniae can ferment inulin (carbohydrate)
 Not usually a primary infection but rather a result of disturbance of o Indicator: Phenol Red
the normal defense barriers o (+) = Yellow [S. pneumoniae ]
o (–) = Red/Pink [Viridans Group]
Predisposing factors:
o Alcoholism  Capsular Swelling Reaction or Nueffeld Quellung Reaction
o Anesthesia o Methylene Blue + Antitoxin + Bacteria
o Malnutrition o Positive: Obvious cell wall [S. pneumoniae ]
o Viral infections of the upper respiratory tract o Negative: No capsule [All other Alpha hemolytic
Streptococci]
Causative agent of Lobar Pneumonia or Pneumococcal Pneumonia:
o Lobar distribution of the infection  Mouse Virulence Test
o Sudden onset with chills, dyspnea and cough o Positive: Death of Test Animal
o Transmitted via aspiration of respiratory secretions o Fred Neufeld

 Pneumonia may be complicated by a pleural effusion that is  Francis Skin Test

usually sterile (empyema) o Test for previous infection
 Also causes bacterial meningitis in all age groups – usually follows o Erythrogenic test
after otitis media or pneumonia o Patient must not have rashes
o Direct smears of CSF – reveal leukocytes and numerous o 2-3 days
gram-positive cocci in pairs o Positive: induration or
o Wheale formation
 Secondary atypical hemolytic uremic syndrome caused by S.
pneumoniae has been reported in children Treatment
 Vaccines are available – heptavalent pneumococcal conjugate
vaccine (PCV7)  Drug of choice: Penicillin
o Part of routine pediatric immunization schedule  Penicillin resistance – Erythromycin or Chloramphenicol
o Recommended for: asplenic individuals, elderly patients
with cardiac or pulmonary disease Viridans Streptococci
Laboratory Diagnosis
 Normal flora of the upper respiratory tract, female genital tract and
gastrointestinal tract
 Gram Stain  Viridans – means “green”
o Gram-positive cocci in pairs (diplococci)  Fastidious, some strain requires CO2 for growth
o Ends of the cells are slightly pointed (oval or lancet
shape)

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 Groups Species Clinical Infections
S. mitis group S. mitis
S. Pneumoniae  Resistant – Enterococci
S. sanguis  Susceptible – Group D Streptococci (Nonenterococci)
S. oralis
Enterococcus
S. mutans group S. mutans
S. sobrinus
 Previously classified as Group D Streptococci
 Natural inhabitants of the intestinal tracts of humans and animals
S. salivarus group S. salivarus
 Common species: E. faecalis, E. faecium
S. vestibularis
 All species produce the cell wall–associated group D antigen in the
Lancefield classification system
S. bovis group S. equinus
S. gallolyticus  Most enterococci are nonhemolytic or α-hemolytic, although some
strains show β-hemolysis
S. infantarius
S. alactolyticus  Sometimes exhibit a pseudocatalase reaction – weak bubbling in
catalase test
S. anginosus group S. anginosus  Ability to grow under extreme conditions: presence of bile, 6.5%
S. constellatus NaCl or alkaline pH
S. intermedius  Can hydrolyze PYR – differentiates them from Group D
Streptococci
Clinical Infections
Clinical Infections
 Are opportunistic pathogens but can, cause disease.
 The most common cause of subacute bacterial endocarditis – a  Frequent cause of nosocomial infection – UTI (most common)
condition associated with a transient bacteremia followed by bacteremia
 Prolonged hospitalization is a risk factor for acquiring enterococcal
 Oral infections such as gingivitis and dental caries (cavities)
bacteremia
 They have also been implicated in meningitis, abscesses,
osteomyelitis, and empyema  Bacteremia: receiving hemodialysis, Immunocompromised patients
with a serious underlying disease, prior surgical procedure
 S. anginosus group – normal flora of oral cavity and  Endocarditis: elderly patients with prosthetic valves or valvular
gastrointestinal tract heart disease
o Associated with abscess formation in the oropharynx,
brain and peritoneal cavity
o S. constellatus subsp. pharyngis – pharyngitis

 S. mitis group – normal flora of oral cavity, gastrointestinal tract

and female genital tract; also transient normal flora of the skin

o Most common isolates associated with bacterial

endocarditis in native valves and less frequently, in
prosthetic valve infections
o S. bovis group – often encountered in blood cultures of
patients with bacteremia, septicemia and endocarditis
 Presence of S. gallolyticus subsp. gallolyticus
in blood cultures – gastrointestinal carcinoma

 S. mutans group – most commonly isolated among the viridans

streptococci
o Usually isolated from the oral cavity
o S. mutans – primary contributor to dental caries
 Most common member of the mutans group
associated with bacteremia

Group D Streptococci
Additional Information
Subdivided into:
 A negative catalyst test result differentiates streptococci and
o Enterococci
enterococci from staphylococci.
o Non-enterococci
 Both groups are nonhemolytic  Weak false positive catalyst reactions can be seen when growth is
 Both groups are positive for Bile Esculin Test taken from media containing blood.
 Facultative Anaerobes grow in the presence of oxygen but are
Tests that differentiate the two groups: unable to use oxygen for respiration
o PYR Test
 Positive: Enterococci Typical gram-positive cell wall consisting:
 Negative: Nonenterococci (Group D 1. Peptidoglycan
Streptococci) 2. Tachoic Acid

o 6.5% NaCl Test  Group A possess the same antigenic c-carbohydrate

 Positive: Enterococci (growth)  Group B have the same c-carbon.
 Negative: Non-enterococci (no growth)
 The M protein molecule causes the streptococcal cell to resist
tragocytosis and plays a role in a adherens of the bacterial cell to
mucosal cell.
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 The M5 protected from infection by streptococcus pyogenes but Samples should be inoculated into selective broth such as:
remains unprotected against infection with the roughly 100 1. 10 microgram/ mL of cholesterol
remaining M protein stereotypes. 2. 15 microgram mL of Thalidoxic Acid

 The M1 stereotype is the most common stereotype seen in The spectrum of infections resembles streptococcus pyogenes and
phagocytosis. includes:
1. Upper respiratory tract infections
2. Skin infection
 The hyaluronic acid capsule streptococcus pyogenes is weakly
3. Soft tissues infection
immunogenic.
4. Invasive infections (Necrotizing Fasciitis)
 The capsule prevents oxidized phagocytosis by neutrophils and  S. Equi Subsp. – animal pathogen rarely isolated from humans
macrophages. o Associated with cases of glomerulonepriritis dramatic
fever.
 The capsule allows the bacterium to mass its antigen and remain
unrecognized by its host.  Optochin is more commonly used procedure to differentiate
streptococcus pneumoniae to the viridans streptococcus.
o Fractal Lysine- two hemolytic exotoxins from
streptococcus  S. anginosus- lancefield group a,c,f,g or n antigen
o Streptolysis O- responsible for hemolysis on SBA plates
incubated and it’s only active in a reduced form.  S. bovic- group D antigen
 Can be measured in the anti-streptolysin O or
ASO Test

o Streptolysis S- slices leukocytes and it is non-

immunogenic

 The four immunologically distinct exotoxin type found in the

streptococcus pyogene which is SPEA

 Hya solubilizes the ground substances of mammalian connective

tissue.

 DNAses B- enzymes are antigenic and antibodies to anase can be

detected after the infection.

 Bacitranin- expense

 CAMP- identified in 1944, known as “CAMP Factor” that acts

synergistically with a metalizing of staphylococcus aureus to cause
enhances lysis of RBC.

 PYR- identification of beta-hemolytic streptococci (diffentiation of

entero from group D)

 Betamaphtalamide is detected in the presence of an anti-dimethyl

amino

 Hippurate Test- used to detect the ability of bacteria to hydrolyze

substrate hippurate into glycine and benzoic acid.

Enzyme: Hippurase (produce the amino acid glycine detected by

the oxidation with Ninhyaan.

 SXT- beta-hemolytic streptococci on blood agar

o The resistance of Sulfamethoxazole is used for the
primary recovery of Group A and B Streptococcus.

 Streptex- rapid latex test system used in the quantitative detection

and identification of the lancefield group of streptococcus.

 Scarlet Fever- 1 to 2 days after bacterial infection is characterized

by a diffuse red rash (5-7 days)

 Mortality Rate reaches greater than 70% if left unthreatened.

 Pneumatic Fever- prophylactic doses of penicillin.

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